Antibacterial properties of green-synthesized noble metal nanoparticles

Antibacterial properties of green-synthesized noble metal nanoparticles

Author's Accepted Manuscript Antibacterial properties of green-synthesized noble metal nanoparticles Jakub Siegel, Kateřina Kolářová, Vladimíra Vosma...

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Author's Accepted Manuscript

Antibacterial properties of green-synthesized noble metal nanoparticles Jakub Siegel, Kateřina Kolářová, Vladimíra Vosmanská, Silvie Rimpelová, Jindřich Leitner, Václav Švorčík

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S0167-577X(13)01282-2 http://dx.doi.org/10.1016/j.matlet.2013.09.047 MLBLUE15815

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Materials Letters

Received date: 20 March 2013 Accepted date: 9 September 2013 Cite this article as: Jakub Siegel, Kateřina Kolářová, Vladimíra Vosmanská, Silvie Rimpelová, Jindřich Leitner, Václav Švorčík, Antibacterial properties of green-synthesized noble metal nanoparticles, Materials Letters, http://dx.doi.org/ 10.1016/j.matlet.2013.09.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antibacterial properties of green-synthesized noble metal nanoparticles Jakub Siegel,a,* Kateina Koláová,a Vladimíra Vosmanská,a Silvie Rimpelová,b Jindich Leitner,a and Václav Švoríka a

Department of Solid State Engineering, Institute of Chemical Technology, 166 28 Prague, Czech

Republic b

Department of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 Prague,

Czech Republic

________________________________________________________________________________ Abstract We report on the antibacterial activity of noble metal nanoparticles prepared by direct sputtering into liquid medium (propane-1,2,3-triol). Silver and gold spherical nanoparticles with diameter of 46 nm (AgNP4-6, AuNP4-6) and gold spherical nanoparticles with diameter of 9-12 nm (AuNP8-12) were investigated. The possibility of managing nanoparticle diameter via controlling temperature of capturing media is also demonstrated. Nanoparticle size and shape were studied by transmission electron microscopy. Optical response of nanoparticle solutions was determined by UV-Vis absorption spectroscopy. Antibacterial properties were tested against two common pollutants (E. coli DBM 3138, a Gram-negative bacteria and S. epidermidis DBM 3179, a Gram-positive bacteria). The cells (concentration of 11,000 cells.ml-1) were incubated together with nanoparticles (concentration 0.8 mg.ml-1). In the presence of silver nanoparticles the growth of E. coli and S. epidermidis was completely inhibited after 24 h. Any growth inhibition of E. coli was observed neither in the presence of smaller nor bigger gold nanoparticles during the whole experiment. Surprisingly, AuNP4-6, but not AuNP8-12 were able to inhibit the growth of S. epidermidis.

Keywords: Gold; silver; nanoparticle; glycerol; antibacterial activity ________________________________________________________________________________ *

Corresponding author:

E-mail address: [email protected] 1

1. Introduction Over the past decade, numerous research studies have demonstrated that the unique electromagnetic, optical, catalytic and bactericidal properties of noble metal nanocrystals are strongly influenced by their shape and size.1-4 Among inorganic antibacterial agents, nanosized silver has been employed most extensively since ancient times to fight infections and control spoilage. The antibacterial and antiviral actions of silver, silver ions, and silver nanoparticles (NPs) have been thoroughly investigated.5-7 In this context, gold has been rarely explored as an antimicrobial agent so far. However, some pioneering works have demonstrated that AuNPs do not show side-effects against eukaryotic cells frequently observed upon usage of Ag NPs.8 When applying metal nanoparticles into living tissues, fundamental attention must be paid to the NPs synthesis process itself. Considering this, direct metal deposition into the glycerol seems to be promising technique combining the advantages of non-toxic and environmentally friendly process completely omitting the usage of solvents or reduction agents compared to classical wetbased methods. Recently, we have demonstrated original approach for noble metal nanoparticle synthesis.9 The novelty of this study lies in the possibility of tailoring the nanoparticle size via controlling the temperature of capturing medium as well as in effort to investigate the functions of nanoparticle (NP) size and metal type (silver and gold) on the antibacterial activity. The antibacterial properties of these NPs were tested against two common pollutants of E. coli (Gram-negative bacteria) and S. epidermidis (Gram-positive bacteria) naturally occurring on the skin and mucous membranes of human.10

2. Experimental 2.1. Materials, apparatus and procedures As a capturing medium for NPs we used anhydrous solution of propane-1,2,3,-triol (Sigma Aldrich, Mw = 92.1 g.mol-1, purity 99.8 %). 3 ml of glycerol were poured into the Petri dish (inner diameter of 4 cm) so that the liquid formed uniform film spread over the dish bottom. Metal sputtering was accomplished in sputtering device SputterCoater SCD 050 (BAL-TEC) using a 99.999 % pure gold and silver target. The sputtering was performed either at room temperature (20 °C) or at lower temperature (5 °C) for deposition time of 300 s, current of 40 mA, total argon pressure of 7 Pa (gas purity 99,99 %), an electrode distance of 5 cm. More detailed description of the deposition procedure can be found in ref.9

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2.2. Analytical methods Colloidal Au and Ag nanoparticle solutions for TEM, UV-Vis analysis and testing of bactericidal activity were prepared from sputtered glycerol media by its dilution with distilled water into the volume of 25 ml. Samples for transmission electron microscopy (TEM, HRTEM) were prepared by putting a drop of the colloidal solution on a copper grid coated with a thin amorphous carbon film placed on filter paper. Excess of solvent was removed. Samples were dried and kept under vacuum in a desiccator before putting them in a specimen holder. TEM characterization of the sample was performed on a JEOL JEM-1010 (JEOL Ltd., Japan) operated at 80 kV. Particle size was measured from the TEM micrographs and calculated by taking at least 100 particles. HRTEM characterization was carried out on JEOL JEM-2200FS (JEOL Ltd., Japan) operated at 220 kV. UV-Vis absorption of NP solutions was carried out on Perkin Elmer Lambda 25 UV-Vis spectrophotometer, equipped with deuterium and halogen lamp light sources. Absorption spectra were collected at room temperature (recording rate 240 nm.min-1, collection interval 1 nm). All spectra were normalized to 1 cm path length of the cuvette. All measurements were baselined on blank sample of corresponding glycerol-water solution.

2.3. Antibacterial tests The antibacterial effect of (Ag,Au)NP4-6 and AuNP9-12 was carried out using two environmental bacterial strains, Gram-negative Escherichia coli (E. coli, DBM 3138) and Gram-positive Staphylococcus epidermidis (S. epidermidis, DBM 3179). Experiments were carried out according to Kolarova et al.11 A single bacterial colonies were grown in Luria–Bertani broth medium (LB) overnight at 37 °C in orbital shaker. The inocula were prepared by serial dilutions of the overnight cultures in fresh physiological saline solution (0.9 % w/v of NaCl). The cells (concentration of 11,000 cells.ml-1) were incubated together with nanoparticles (concentration 0.8 mg.ml-1). In parallel, E. coli and S. epidermidis incubated in glycerol and in saline solution were used as two controls. All samples were incubated statically at 24°C for 6 and 24 h. Aliquots of 25 μl from all samples were subsequently placed on LB agar plates. The growth of E. coli and S. epidermidis was evaluated after 24, 30 and 48 h. Each sample was done separately in triplicate.

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3. Results and Discussion 3.1 Nanoparticle size control and apportion spectra Quantitatively, the control of nanoparticle size based on the capturing media temperature may be expressed upon a simple, generally known approach12. Nanoparticles growth is supposed to be governed by diffusion from the bulk of colloidal dispersion to the solution/particles interphase. Considering the Fick’s law for diffusion flux of atoms (source atomic clusters) and the geometry of the system it follows for the radius of nanoparticle, dr dt

Vm D Cbulk  Csurf r



(i)

where Vm is molar volume of particle material, D is diffusion coefficient and Cbulk, Csurf stand for concentration of particle material in the bulk and on the surface of the nanoparticle, respectively. It follows from Eq. (i) that the temperature dependence of particles growth rate is predominantly given by temperature dependence of diffusion coefficient D. According to the Stokes-Einstein relation13 the diffusion coefficient increases with temperature, accelerating the particles growth rate, which results in considerably larger particles (see Fig. 1b,c). UV-Vis absorption spectra of metal NP solutions are shown in Fig. 2. Both AgNP4-6 and AuNP4-6 exhibit distinctive narrow peaks with maximum absorption at 400 and 520 nm, respectively. These spectra are almost identical to those obtained in our former study on Ag and Au with average diameter of 3-5 nm indicating stable solutions with minimal particle dispersion.9 Considerable broadening with pronounced red shift of absorption peek occurs at AuNP9-12. This phenomenon originates from both, increase in dimensions of individual Au particles and broader size distribution compared to AuNP4-6. Distinctive red coloration of AuNP4-6 turns into purple one, belonging to AuNP9-12, as the average size increases from ca 5 to 10 nm.

3.2 Antibacterial properties As we have expected, the growth of E. coli and S. epidermidis was completely inhibited in the presence of AgNP4-6 after 24 h (for both 6 and 24 h incubated samples) when compared to the control samples (bacteria incubated in glycerol or physiological saline solution [PBS]), see Tab. 1. The growth inhibition of both bacterial strains was further maintained even after 30 and 48 h of growth, thus suggesting strong bactericidal activity of AgNP4-6. On contrary, we observed any growth inhibition of E. coli neither in the presence of AuNP4-6, nor AuNP9-12 during the whole experiment. Surprisingly, AuNP4-6, but not AuNP9-12, were able to inhibit the growth of S. epidermidis and the effect was preserved for whole tested period (48 h), when compared to control samples. 4

The antibacterial action of Ag+ ions has been broadly reported so far,14 also AgNPs repeatedly showed their potency against bacteria.15 Thus, our data (growth inhibition of both bacterial strains by AgNP4-6) is in agreement with other groups results. Biocidal properties of AuNPs of similar size (5 nm) as prepared by our group (4-6 nm), were observed by Lima et al.16 Their AuNPs dispersed on zeolites were interestingly effective against Gram-negative E. coli and S. typhi (90-95% growth inhibition). It has been reported, that the antibacterial activity of Ag NP is dependent on particle size and shape.3,7 Thus, it is very likely that also the size of Au NP plays significant role in the antimicrobial action.

4. Conclusions In summary, novel technique for the size-controlled synthesis of noble-metal nanoparticles is described and experimentally examined. The method is based on temperature control of capturing media during the sputtering deposition process into propane-1,2,3-triol. In this way the size of Au nanoparticles can be varied from ca 4-12 nm. Bactericidal action of such synthesized NPs was examined against two common pollutants (E. coli and S. epidermis). While AgNP4-6 completely inhibited both bacteria strains after 24h, AuNPs exhibited pronounced inhibition selectivity regarding to the specific NP dimensions. Regardless of AuNP size, any growth inhibition of E. coli occurred during the whole experiment. Contrary to that, AuNP4-6 were able to inhibit the growth of S. epidermidis.

Acknowledgements Financial support of this work from the GACR projects No. P108/11/P337 and P108/10/1106 is gratefully acknowledged.

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References 1. Burda C, Chen X, Narayanan R, El-Sayed MA. Chemistry and properties of nanocrystals of different shapes. Chem Rev 2005;105:1025-102. 2. Žvátora P, ezanka P, Prokopec V, Siegel J, Švorík V, Král V. Polytetrafluorethylene-Au as a Substrate for Surface Enhanced Raman Spectroscopy. Nanoscale Res Let 2011;6:366. 3. Pal S, Tak YK, Song JM. Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Appl Environ Microbiol 2007;73:1712-20. 4. Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT, Yacaman MJ. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:2346-53. 5. Chou WL, Yu DG, Yang MC. The preparation and characterization of silver-loading cellulose acetate hollow fiber membrane for water treatment. Polym Adv Technol 2005;16:600-7. 6. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TM, Kim JO. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 2000;52:662–8. 7. Panáek A, Kvítek L, Prucek R, Kolá M, Veeová R, Pizúrová N, Sharma VK, Nevná T, Zboil R. Silver Colloid Nanoparticles: Synthesis, Characterization, and Their Antibacterial Activity. J Phys Chem B 2006;110:16248-53. 8. White JML, Powell AM, Brady K, Russell-Jones, R. Severe generalized argyria secondary to ingestion of colloidal silver protein. Clin Exp Dermatol 2003;28:254-6. 9. Siegel J, Kvítek O, Ulbrich P, Kolská Z, Slepika P,Švorík V. Progressive approach for metal nanoparticle synthesis, Mater Lett 2012;89:47-50. 10. Lowy DF. Staphylococcus aureus infections. N Engl J Med 1998;339:520–532. 11. Kolarova K, Vosmanska V, Rimpelova S, Svorcik V. Effect of plasma treatment on cellulose fiber. Cellulose 2013;20:953-61. 12. Park J, Joo J, Kwon SG, Jang Y, Hyeon T. Synthesis of Monodisperse Spherical Nanocrystals. Angew Chem Int Ed 2007;46:4630-60. 13. Kholodenko AL, Douglas JF. Generalized Stokes-Einstein equation for spherical particle suspensions. Phys Rev E 1995;51:1081-90. 14. Klaus T, Joerger R, Olsson E, Granqvist CG: Silver based crystalline nanoparticles, microbially fabricated. Proc Natl Acad Sci USA 1999;96:13611–4. 15. Raffi M, Hussain F, Bhatti TM, Akhter JI, Hameed A, Hasan MM: Antibacterial characterization of silver nanoparticles against E. coli ATCC-15224. J Mater Sci Technol 2008;24:192–6. 6

16. Lima E., Guerra R., Lara V., Guzmán A. Gold nanoparticles as efficient antimicrobial agents for Escherichia coli and Salmonella typhi, Chemistry Central Journal 2013;7:11.

Table Tab. 1 Inhibitory effect of silver and gold nanoparticles against Gram-negative (E. coli) and Gram-positive (S. epidermidis) bacteria (atime length of bacterial growth on LB plates after inoculation, ban empty circle indicate inhibition effect, ca full circle indicate positive growth, dphysiological saline solution, e

a half full circle indicate 50 % growth).

E. coli

Bacteria Growth time (h)

a

S. epidermidis

24

30

48

24

30

48

Ag NP4-6

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Au NP4-6

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Au NP9-12

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z

z

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z

Glycerol

z

z

z

{

z

z

PBSd

z

z

z

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z

z

c

Figure captions Fig. 1 TEM images of nanoparticles of different composition and size AgNP4-6 (a), AuNP4-6 (b), and AuNP9-12 (c) together with HRTEM image of corresponding area (d). Indexes refer to NP diameter in nm. Fig. 2 UV-Vis absorption spectra of Ag (AgNP4-6) and Au (AuNP4-6, AuNP9-12) aqueous solutions of different nanoparticle size (indexes refer to average NP diameter in nm). Fig. 3 Photograph showing the inhibition effect of AgNP4-6 on S. epidermidis (a) positive control of bacterial colonies growing on agar plate, (b) bacterial sample treated with silver nanoparticles.

Highlights Ag and Au nanoparticles were prepared by sputtering into propane-1,2,3-triol. Au nanoparticle size differed depending on the temperature of capturing media. Average Au nanoparticle diameter varied from 5-11 nm. Antibacterial properties of nanoparticles were tested against E. coli and S. epidermidis. Silver nanoparticles completely inhibited the growth of both pollutants. 7

Fig. 1

b

a

25 nm

25 nm

d

c

25 nm

4 nm

Fig. 2 8

Siegel et al., Antibacterial properties of green-synthesized noble metal nanoparticles

a

b

Fig. 3

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